The present invention generally relates to tissue engineering applications, and more particularly for repair of cartilage and treatment of bony defects by uses of chondrocyte-like cells prepared from fibroblasts.
Annually, over one million procedures involving cartilage replacement are performed in the United States. Many are as a result of debilitating ailments affecting articular cartilage. Articular cartilage is a thin layer of soft connective tissue (0.5-5 mm thick) that covers the articulating surfaces of long bones in synovial joints. The principal function of articular cartilage is to redistribute applied loads and to provide a low friction-bearing surface to facilitate movement within these joints. The most common of the pathological conditions affecting articular cartilage is osteoarthritis (OA), a degenerative joint disease that afflicts between 32-38 million Americans. OA is characterized by a progressive loss of cartilage tissue due to excessive mechanical trauma or to continual loading overtime, resulting in joint pain and stiffness. Unlike other connective tissues, cartilage has a limited reparative ability because it lacks a reservoir of undifferentiated mesenchymal cells that can be recruited to a defect in aid in wound repair (whereas bone possesses marrow and periosteum-derived precursor cells on the inner and outer portions of the tissue, respectively). In addition, cartilage is an avascular tissue, and therefore, cannot rely upon the circulatory system to transport nutrients and cells to sites of damage. Thus, one approach that has been used has been to expose damaged cartilage tissue to stimuli by drilling or scraping through the cartilage into the subchondral bone to cause bleeding. However, when repair does occur, it often results in the formation of fibrocartilage which lacks the structural components and organization to withstand the mechanical demands of the natural tissue. As such, there is a need to develop effective replacement therapies for articular cartilage to restore its biological function.
Current surgical procedures to correct cartilage tissue defects involve primarily the use of autogenous or allogeneic tissue grafts. Autogenous grafts are biocompatible, but their use is limited due to a lack of tissue supply, and because of pain and morbidity which often develop at the donor site. Furthermore, the mechanical integrity of the tissue at the donor site may be compromised, rendering the remaining cartilage more susceptible to damage in the future. Allograft tissue, on the other hand, is more readily available, but the risk of disease transmission and immune responses to alloantigens present difficulties.
Recent advances in the fields of cell and molecular biology, biotechnology, and biomaterials have led to the emergence of tissue engineering, an exciting new discipline applying both engineering and life science principles to the formation of biological substrates capable of regenerating functional mammalian tissues both in vitro and iii vivo. Present attempts to engineer articular cartilage involve the isolation of primary, differentiated cells (i.e. chondrocytes) from biopsies of existing cartilage tissue and seeding these cells onto three-dimensional carrier materials. A major limitation of these techniques is that the cells are often procured from an autologous source. As such, the problem of limited donor tissue supply is not circumvented as the cellular component of the implant is harvested from host cartilage tissue. In addition, invasive surgical procedures are required to obtain the necessary quantity of cells. Alternatively, the procurement of cells from cadavers carries the inherent risk of transfer of pathogens, and the undue expense of screening for the presence of harmful pathological agents. The drawback to both of these approaches stems from the source of cellular material.
It is known that connective-tissue cells, including fibroblasts, cartilage cells, and bone cells, can undergo radical changes of character. Thus, as explained by Alberts et al., Molecular Biology of the Cell, (2nd Ed., 1989, pp. 987-988), a preparation of bone matrix may be implanted in the dermal layer of the skin and some of the cells there converted into cartilage cells and later others into bone cells. Cultured cartilage cells, or chondrocytes, can be converted so as to acquire characteristics of fibroblasts and stop producing type II collagen (characteristic of cartilage), but instead start producing type I collagen (characteristic of fibroblasts).
Hunziker, U.S. Pat. No. 5,368,858, issued November 29, 1994, describes matrix compositions for the treatment or repair of cartilage including a transforming growth factor associated with a delivery system contained within the matrix. The transforming growth factor is described as TGF-xcex2 said to be capable of inducing conversion of repair cells into chondrocytes. Shorts, U.S. Pat. No. 5,632,745, issued May 27, 1997, describes a cartilage repair unit with a matrix in which xe2x80x9crepair factorsxe2x80x9d such as fibroblast growth factor and xe2x80x9cTGF-xcex2xe2x80x9d are included. Thus, a chondrogenic growth-supporting matrix is said to permit vascular invasion and cellular migration between healthy cancellous bone and the damaged articular cartilage area.
A great variety of materials said to be useful as scaffolds, or matrices, are known and proposed for cartilage implants. For example, materials such as collagen gels, poly(D,L-lactide-co-glycolide (PLGA) fiber matrices, polyglactin fibers, calcium alginate gels, polyglycolic acid (PGA) meshes, and other polyesters such as poly-(L-lactic acid) (PLLA) and polyanhydrides are among those suggested and are in varying degrees of development and use. However, osteoinductive cells, such as improved chondrocyte-like cells for tissue engineering applications, without the problems of invasive surgery for the recipient from other regions of the body or cells without risk of disease transmission from others, remain desirable for uses such as seeding the synthetic matrices.
In one aspect of the present invention, fibroblasts are first converted to chondrocyte-like cells by treating with a combination of agents. One agent is an hypoxia mimicking agent and the second agent is a chemical inhibitor of protein kinase C (PKC). These converted chondrocyte-like cells display at least one cartilage phenotype.
Typical cartilage phenotypes are the cellular morphology, the production of cartilage matrix components, the expression of the aggrecan and type II collagen MRNA, and the down-regulation of type I collagen mRNA. Thus, the converted chondrocyte-like cells of the invention will typically show an increase in aggrecan and in type II collagen mRNA expressions and a decrease in type I collagen MRNA. Such a display of cartilage phenotype is illustrated, for example, by FIG. 5.
The initial conversion of chondrocyte-like cells is preferably where the cells are cultured in a high density micromass with conversion occurring in a period occurring from about 12 hours up to about 36 hours. The high density micromass culture is preferably achieved on a biocompatible (preferably biodegradable) scaffold. Such a scaffold assists in creating the high density micromass and can subsequently serve as a template for cartilage repair. The so-converted cells, when attached to a scaffold, can thereafter be maintained with the chondrocyte phenotype for about one to two weeks prior to implantation by maintaining them in a chemically defined, serum-free media.
The inventive cells are useful in a variety of reparative applications, including the repair of articular cartilage in synovial joints, of tracheal and esophageal cartilage, and for cosmetic purposes including modification of nasoseptal and ear cartilage. Other applications include intervertebral disc repair as an alternative to spinal fusion following disc herniation, oral and maxillofacial surgery including temporomandibularjoint disc replacement, and treatment of osteochondral and bony defects.
Chondrocyte-like cells of the invention are preferably used in reparative applications by being placed in a suitable matrix in the desired site for repair. The matrix is preferably partly or entirely formed by scaffold on which the cells were preferably seeded and converted. Preferred matrices are biodegradable, and can direct cellular remodeling through mechanical influences such as fine fibers and surface treatments to enhance cell attachment and alignment.